To expand our biological atomic force (Bio-AFM) technology, instrumentation and data analysis methods, we have made progress with our Raman-AFM Instrumentation and have initiated a new Quartz Crystal Microbalance-Dissipation (QCM-D)and AFM instrumentation effort. Applying these expanding technology and sharing an existing Bio-AFM facility, we have advanced a number of biomedical investigations in collaboration with NIH intramural and extramural researchers. Major sub-projects with notable results include: (1) We have investigated the macromolecular structure and nanomechanical properties of five malaria vaccine candidates via Bio-AFM and related bioanalysis in collaboration with Dr. David Narum (NIAID, NIH), Dr. Louis Miller(NIAID, NIH), Dr. Patrick Duffy and other co-investigators at the Laboratory of Malaria Immunology and Vaccinology, NIAID, NIH). These protein antigens for malaria vaccine are being produced via recombinant-protein biotechnology, purified, and characterized in a manner suitable for human trials and scale-up productions. We have focused on using AFM, QCM-D, and related studies to understand the structural properties of the developing vaccine products, including most recently an rEPA-pfs25 conjugate and Qbeta virus-like particles, under a range of fluid and surface conditions. Our results have implications for malaria biology and human immunological response. This collaboration has contributed to two publications, many presentations, and several manuscripts in preparation. (2) We have continued our AFM studies of clathrin and clathrin coated vesicles (CCVs) in collaboration with Dr. Ralph Nossal (NICHD, NIH), Prof. Eileen Lafer (Univ. Texas Health Sciences Center, San Antonio), and other coworkers. Clathrin triskelia form the outer clathrin lattice cages of the CCVs during subcellular trafficking via interactions with adaptor proteins, membrane lipids, and other cofactors. The intricacies of these dynamic macromolecular constructs have inspired numerous structural and functional studies. We have published a study on resolving variable profiles of triskelia on mica surfaces by AFM at a resolution comparable to electron microscopy. Classical tri-leg, filamentous pin-wheel shapes, as well as non-planar triskelion conformations and triskelion-triskelion dimers, are readily observed both dried on mica surface and under buffers. Pentagonal and hexagonal lattice structures are well visualized in a variety of clathrin assemblies with or without AP180 adaptors, similar to those of the native CCVs purified from bovine brains. We have also produced single molecule force spectroscopy (SMFS) of triskelia and CCVs under buffers and revealed, also for the first time, a series of internal energetic barriers that characterize triskelion heavy chain folding and unfolding, including molecular sequence and structure periodicity for both the seven repeating 145aa motifs and numerous 30aa hairpins. The dynamic stability of these domains has been obtained. The addition of QCM-D technology has enhanced the accuracy of our nanomechnical characterization for clathrin and their assembled structures. (3) We have explored a number of additional intramural and extramural collaborations in such areas as developing AFM-related nanotechnology;characterizing structure and function of live cells, G-protein coupled receptor (GPCR), mycobacterium tuberculosis membrane, bacterial bio-films, polymer and biomimicking materials;and advancing fundamental surface and material sciences.